α‐Helix‐to‐random‐coil transitions of two‐chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross‐linked selectively at C‐190

Abstract

SynopsisA method is described for preparation of a species of 88 tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomywin, comprising aa and a8 species, is oxidized with ferricyanide, &sulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the 8-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (D"), unfolded in urea, and a and 8 chains separated by ionexchange chromatography. (4) The C36-blocked 8 chains are refolded by dialysis. (5) The refolded, C36-blocked 88 species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, ClSO-cross-linked 88 product is separated from contaminating species-mostly completely blocked 8-chains and multichain cross-linked molecules-by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaD&O4/poIyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked 88 species and for its D"-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked 88 species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of Cl90-cross-linked aa tropomydn. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) 88 species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. I t is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the pbysics. tion of particular local conformational transitions in various biological processes in the muscle tissue in which these proteins reside?-" Their struc-'To whom reprint requests should be a d d r d .

Electrostatic Interactions Control the Parallel and Antiparallel Orientation of .alpha.-Helical Chains in Two-Stranded .alpha.-Helical Coiled-Coils

“…On this basis, Holtzer and co-workers have proposed the "continuum-of-states" theory (Skolnick & Holtzer, 1986), suggesting that "a broad spectrum of molecular states are allowed" between the coiled-coil form and the unfolded state. This theory had been used to explain the differences in the shapes of temperature denaturation curves of a variety of cross-linked and non-cross-linked tropomyosins (Skolnick & Holtzer, 1986;Holtzer et al, 1990;Bracken et al, 1988). Whether the denaturation proceeds in a two-state or a multi-state fashion will have no effect on the interpretation of our results.…”

“…However, it cannot be assumed that all coiled-coils follow a two-state denaturation since the transition from coiled-coil to random coil appears to be very fast (Mo et al, 1993) and any possible intermediates may not be easily detectable by conventional CD spectroscopy. In addition, other native coiled-coils appear to exhibit biphasic denaturation curves (Lehrer, 1978;Lehrer et al, 1989;Lehrer & Qian, 1990; Lehrer & Stafford, 1991;Bracken et al, 1988; Greenfield & Hitchcock-DeGregori, 1993), suggesting the presence of folded intermediate states. On this basis, Holtzer and co-workers have proposed the "continuum-of-states" theory (Skolnick & Holtzer, 1986), suggesting that "a broad spectrum of molecular states are allowed" between the coiled-coil form and the unfolded state.…”

Design and Characterization of an Intramolecular Antiparallel Coiled Coil Peptide

“…As shown in Figure 7, the intensity of the CD signal for the CCSL peptide decreased as the urea concentration was increased, reflecting the loss of a-helical structure. For simplicity the urea denaturation curve was analyzed by assuming a two-state folding/unfolding transition (Pace, 1986), with an awareness that a more complex unfolding equilibrium may exist (Bracken et al, 1988). The values for the transition region were obtained by extrapolating from the linear portions of the denaturation curve at low and high denaturant concentrations (Pace, 1986).…”

Kinetics of self-assembly of alpha alpha-tropomyosin coiled coils from unfolded chains.

“…Many physical studies of thermally or denaturant-induced unfolding establish the equilibrium helix content of particular coiled coils (4)(5)(6)(7)(8)(9)(10)(11). Nevertheless, important questions remain concerning the contribution of intra-vs. interchain interactions, hydrophobic vs. electrostatic forces, and the number and nature of populated conformational states at equilibrium (1,7).…”